Methods for estimating prion concentration in fluids and tissue by quantitative pmca

ABSTRACT

The present embodiments disclose methods for estimating PrP Sc  concentration in fluids and tissues by quantitative PMCA.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Patent Application No. 61/345,940, filed May 18, 2010, and U.S. Provisional Patent Application No. 61/345,760, filed May 18, 2010, both of which are incorporated by reference herein in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under R01NS049173 and P01AI077774 awarded by the National Institutes of Health. The Government has certain rights in the invention.

BACKGROUND

Transmissible spongiform encephalopathies (TSE), also known as prion diseases, are a group of neurodegenerative diseases that affect humans and animals. Creutzfeldt-Jakob disease (CJD), kuru, Gerstmann-Straussler-Scheiker diseases (GSS), and fatal familial insomnia (FFI) in humans, as well as scrapie and bovine spongiform encephalopathy (BSE) in animals, are examples of TSE diseases.

It is known that a key characteristic and marker of prion diseases is the formation of an abnormally shaped protein named PrP^(Sc). However, prion diseases are characterized by an extremely long incubation period. Thus, concentrations of PrP^(Sc) are at low levels for a long period of time. As such, one important objective of prion research has been to detect small amounts of PrP^(Sc) in diverse samples.

PrP^(Sc) is a post-translationally modified version of a normal protein, termed PrP^(C). PrP^(C) is found naturally on the membranes of animal and human cells. The infective unit of PrP^(Sc) is understood to be a β-sheet rich oligomeric structure, which converts PrP^(C) to PrP^(Sc) by integrating PrP^(C) into a growing aggregate (FIG. 1). In light of this replication model, it has been found that PrP^(Sc) can be detected with high sensitivity by protein misfolding cyclic amplification (PMCA). See U.S. Pat. No. 7,351,526 and U.S. Patent Application Pub. No. 2006/0263767, each of which is incorporated by reference herein it its entirety. In the context of prion diseases, PMCA-based PrP^(Sc) detection typically involves: (i) contacting a sample with PrP^(C) (e.g., by contacting a suspected diseased sample with a tissue homogenate containing PrP^(C)); (ia) incubating the sample/PrP^(C) mixture; (ii) disagreggating any aggregates formed during step (ia) (e.g., by sonication); (iii) repeating steps (ia) and (ii) a plurality of times; and (iv) detecting the presence of PrP^(Sc) within the sample (e.g., by western blotting). See FIG. 2.

What is still needed in the art, however, is a quantitative procedure for determining the concentration—rather than merely detecting the presence—of PrP^(Sc) in fluids and tissues. The present embodiments disclose such a procedure.

SUMMARY

In one embodiment, a method for estimating the concentration of PrP^(Sc) in a sample is provided.

In one aspect of the method, a calibration curve is provided, by:

preparing a plurality of stock solutions, each having a known concentration of PrP^(Sc);

separately mixing each of the stock solutions with a first PrP^(C) source to form separate stock reaction mixes;

performing a plurality of protein misfolding cyclic amplification cycles on the separate stock reaction mixes, each cycle comprising:

-   -   incubating the stock reaction mix; and     -   disrupting the stock reaction mix;

subjecting the separate amplified stock reaction mixes to an assay after each cycle, until a prion signal is detected;

comparing the concentration of each stock solution with the number of cycles required to detect the prion signal; and

plotting the comparison in the form of a standard calibration curve.

In another aspect of the method, the calibration curve is used to estimate the concentration of PrP^(Sc) in the sample, by:

mixing the sample with a second PrP^(C) source to form a sample reaction mix;

performing a plurality of protein misfolding cyclic amplification cycles on the sample reaction mix, each cycle comprising:

-   -   incubating the sample reaction mix; and     -   disrupting the sample reaction mix;

subjecting the amplified sample reaction mix to an assay after each cycle, until a PrP^(Sc) signal is detected; and

comparing the number of cycles required to detect the PrP^(Sc) signal to the calibration curve.

In another embodiment, a method for estimating the concentration of prion in a sample is provided, the method comprising:

mixing the sample with a non-pathogenic protein to form a reaction mix;

performing a plurality of protein misfolding cyclic amplification cycles on the reaction mix, each cycle comprising:

-   -   incubating the reaction mix; and     -   disrupting the reaction mix;

subjecting the amplified reaction mix to an assay after each cycle, until a prion signal is detected; and

comparing the number of cycles required to detect the prion signal to a predetermined calibration curve.

In another embodiment, a kit for detecting and quantifying prion in a sample is provided, the kit comprising:

(a) a non-pathogenic protein;

(b) a sonicator; and

(c) a calibration curve.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying figures, which are incorporated in and constitute a part of the specification, illustrate various methods, results, and so on, and are used merely to illustrate various example embodiments.

FIG. 1 illustrates an example schematic representation of the conversion of PrP^(C) to

FIG. 2 illustrates an example diagrammatic representation of the protein misfolding amplification procedure.

FIG. 3 a illustrates western blot assays (3F4 antibody) of stock solutions of PrP^(Sc) of various concentrations, upon being subjected to normal brain homogenate and serial rounds of PMCA (144 cycles).

FIG. 3 b illustrates a calibration curve, based on a plot of PrP^(Sc) concentration vs. the number of PMCA rounds required to detect PrP^(Sc) by western blot assay.

FIG. 4 is a flow chart of an example method for estimating the concentration of prion in a sample, where a predetermined calibration curve is provided.

FIG. 5 illustrates western blot assays of PrP^(Sc)-affected hamster spleen suspended in normal hamster brain homogenate and subjected to serial PMCA, as compared to various control samples.

FIG. 6 illustrates western blot assays of PrP^(Sc)-affected hamster spleen suspended in normal hamster brain homogenate and subjected to serial PMCA. The samples were taken at various time periods after the hamsters were inoculated with PrP^(Sc).

FIG. 7 illustrates plots of concentrations of PrP^(Sc) in PrP^(Sc)-affected hamster spleen, brain, buffy coat, and plasma after serial rounds of PMCA. The samples were taken at various time periods after the hamsters were inoculated with PrP^(Sc).

DETAILED DESCRIPTION

The present embodiments disclose a method for estimating PrP^(Sc) concentration in fluids and tissues by quantitative PMCA.

In one embodiment, a calibration curve is determined. For example, a plurality of stock solutions is prepared, each having a known concentration of PrP^(Sc). The stock solutions are separately mixed with a first PrP^(C) source to form separate stock reaction mixes. The stock reaction mixes are incubated and subjected to sonication. The incubation and sonication steps may be repeated a plurality of times, until a PrP^(Sc) signal is detected (by, e.g., western blotting) for each stock reaction mix. The concentration of each stock solution is compared with the number of times the incubation and sonication steps were repeated, to determine a calibration curve.

In one particular embodiment, PrP^(Sc) was partially purified by precipitation in the presence of sarkosyl. This partially purified PrP^(Sc) was used as a stock solution in buffer. To estimate the PrP^(Sc) concentration in the stock solution, partially purified PrP^(Sc) in the stock solution was subjected to deglycosylation and, subsequently, to western blot assay. To determine the PrP^(Sc) concentration of the stock solution, the stock solution western blot assay signal was compared to western blot and enzyme-linked immunosorbent assay signals of known concentrations of PrP^(Sc).

Once the PrP^(Sc) concentration of the stock solution was estimated, the stock solution was diluted and separated into several sub-solutions having various known PrP^(Sc) concentrations ranging from 1×10⁻⁸ to 1×10⁻¹⁹ g. The sub-solutions were separately spiked into separate normal hamster brain homogenates to form stock reaction mixes. The stock reaction mixes were subjected to serial rounds of PMCA cycles. In one particular embodiment, one “round” of PMCA cycles corresponded to 144 cycles. More or fewer than 144 cycles may also be used. The number of PMCA rounds required to produce a signal detectable by western blot was determined.

FIG. 3 a illustrates western blot assays (3F4 antibody) of the stock reaction mixes. All samples except the normal brain homogenate (NBH) used as a migration control were digested with proteinase K (PK).

The concentrations of the sub-solutions were plotted against the number of PMCA rounds required for detection, to provide a calibration curve. The results are shown in FIG. 3 b. More particularly, the western blots from FIG. 3 a were analyzed by densitometry, and the last detectable signal after each PMCA round was plotted, yielding a standard calibration curve to estimate PrP^(Sc) concentrations.

It was determined that there is a direct relationship between the quantity of PrP^(Sc) in a given sample and the number of PMCA cycles necessary for its detection. By extrapolating the number of PMCA rounds required to detect an unknown sample, the concentration of PrP^(Sc) in the sample may be estimated.

Thus, in one embodiment, an unknown sample may be subjected to a PrP^(C) source to form a sample reaction mix. The sample reaction mix is incubated and subjected to sonication. The incubation and sonication steps may be repeated a plurality of times, until a PrP^(Sc) signal is detected for the sample reaction mix. The number of times the incubation and sonication steps were repeated is compared to the predetermined calibration curve to determine the concentration of PrP^(Sc) in the sample.

In one embodiment, as depicted in FIG. 4, where a calibration curve is known and provided, e.g., as a part of a kit, a method 400 for estimating the concentration of prion in a sample may comprise:

mixing the sample with a non-pathogenic protein to form a reaction mix (step 410);

performing a plurality of protein misfolding cyclic amplification cycles on the reaction mix (step 420), each cycle comprising:

-   -   incubating the reaction mix (sub-step 420 a); and     -   disrupting the reaction mix (sub-step 420 b);

subjecting the amplified reaction mix to an assay after each cycle, until a prion signal is detected (step 430); and

comparing the number of cycles required to detect the prion signal to a predetermined calibration curve (step 440).

In one embodiment, a kit for detecting and quantifying prion in a sample is provided, the kit comprising:

(a) a non-pathogenic protein source;

(b) a sonicator; and

(c) a calibration curve.

EXAMPLES Example 1 Sample Preparation

Syrian hamsters were intraperitoneally inoculated with 263,000 prions and monitored for the appearance of clinical symptoms, using a standard scale known in the art. When disease was confirmed, urine was collected using metabolic cages. The hamsters were then killed by CO₂ inhalation, and brains, spleens, and blood were collected.

Brain and spleen homogenates were prepared at 10% (wt/vol) in PBS plus Complete cocktail of protease inhibitors (Boehringer Mannheim). The samples were clarified by a 45 s low speed centrifugation. Blood samples were obtained directly from the heart in tubes containing citrate. Plasma and buffy coat were separated by centrifugation in ficoll gradient. Samples of normal brain homogenate used for PMCA substrate were obtained after perfusing hamsters with PBS and 5 mM EDTA. Solutions of 10% normal brain homogenate were made in conversion buffer (PBS without Ca²⁺ and Mg²⁺ with 150 mM NaCl, 1.0% triton X-100, and Complete protease inhibitors). Debris was removed by a 45 s low speed centrifugation in an Eppendorf centrifuge.

Example 2 PrP^(Sc) Partial Purification by Sarkosyl Precipitation

To minimize interference in PMCA from other components present in tissues and fluids, PrP^(Sc) was partially enriched by sarkosyl precipitation. More particularly, samples were incubated with one volume of 20% sarkosyl for 10 min at room temperature and centrifuged at 100,000 g for 1 h at 4° C. Supernatants were discarded and pellets were re-suspended into two volumes of 10% sarkosyl. The centrifugation process was repeated, and pellets were re-suspended directly in 10% normal brain homogenate prepared in conversion buffer. Following this protocol, PrP^(Sc) was recovered in the pellet fraction at greater than 90% yield.

Example 3 PMCA Procedure

Samples were loaded onto 0.2 mL PCR tubes. Tubes were positioned on an adaptor placed on a plate holder of a microsonicator (Misonix model 4000), and samples were subjected to cycles of 30 min incubation at 37° C., followed by a 20 s pulse of sonication set at a potency of 7.5 (75%). Samples were incubated, without shaking, immersed in the water of the sonicator bath. Standard PMCA rounds included 144 cycles. After each round of cycles, a 10 μL aliquot of the amplified material was diluted into 90 μL of normal brain homogenate and a new round of PMCA cycles was performed.

Example 4 PrP^(Sc) Detection

Samples were digested with 50 μg mL⁻¹ of PK at 37° C. for 1 h, and the reaction was stopped by adding NuPAGE LDS sample buffer. The proteins were fractionated using 4-12% SDS-PAGE, electroblotted into Hybond ECL nitrocellulose membrane, and probed with the 3F4 antibody (Covance) (dilution 1:5,000). The immunoreactive bands were visualized by ECL Plus western blotting detection system and quantified by densitometry using a UVP Bioimaging System EC3 apparatus.

Example 5 Detection of PrP^(Sc) in the Spleen of Scrapie-Affected Hamsters

As described in Example 1, samples of brain, spleen, blood, and urine were collected from five hamsters exhibiting clinical signs of disease after intraperitoneal inoculation with 263,000 prions. As described in Example 2, the PrP^(Sc) was partially purified by sarkosyl precipitation to remove components that may affect PMCA efficiency. After centrifugation, PrP^(Sc) pellets were re-suspended directly into healthy hamster brain homogenate and subjected to serial rounds of 144 PMCA cycles.

Three spleen samples were positive for prion disease. Of those three, PrP^(Sc) was detectable after two rounds of PMCA for two samples and after the third round for the third sample. FIG. 5 illustrates western blot assays of PrP^(Sc)-affected hamster spleen suspended in normal hamster brain homogenate and subjected to serial PMCA. The three scrapie spleen samples are labeled SS1, SS2, and SS3.

With further reference to FIG. 5, control samples of normal (i.e., non-infected) spleen homogenate (samples NS1-NS6) and brain homogenate (samples NB1-NB4) were subjected to the same PMCA procedure to assess the rate of spontaneous appearance of PrP^(Sc) reactivity. Normal brain homogenate (NBH) not digested with PK was used as a migration control. No PrP^(Sc) signal was detected after six rounds of PMCA in any of the control samples.

Extrapolation from the calibration curve of FIG. 3 b provides that the average concentration of PrP^(Sc) in the symptomatic spleen was 20 pg g⁻¹. PrP^(Sc) concentrations in other tissues and fluids were also analyzed. The results are shown in Table 1:

TABLE 1 PrP^(Sc) Concentration in Scrapie-Affected Hampsters Source PrP^(Sc) Concentration in Tissues (g/g) and fluids (g/mL) Brain  2.3 × 10⁻⁵ ± 6.8 × 10⁻⁶ Spleen 2.0 × 10⁻¹¹ ± 1.1 × 10⁻¹¹ Buffy Coat 2.6 × 10⁻¹³ ± 2.4 × 10⁻¹³ Plasma 1.3 × 10⁻¹⁴ ± 1.1 × 10⁻¹⁴ Urine 2.0 × 10⁻¹⁶ ± 1.7 × 10⁻¹⁶

Example 6 Dynamic Distribution and Quantification of PrP^(Sc) in Different Tissues and Fluids

To evaluate the application of quantitative PMCA to determine the concentration of prions in various tissues and fluids, and to understand the dynamic of PrP^(Sc) formation and accumulation in tissues and fluids at distinct stages of the disease, PrP^(Sc) levels in brains, spleens, blood fractions (plasma and buffy coat), and urine were measured at different time periods after infection.

Specifically, tissue extracts were obtained from hamsters intraperitoneally infected with 263,000 prions. Animals were sacrificed at the following time periods: 0, 2, 4, 9, 14, 21, 30, 43, 50, 71, 81, and 110 days post-inoculation. Under these conditions, animals showed the disease symptoms an average 110 days after inoculation. Samples from each of the tissues at each of the times from five different animals per group were suspended in normal hamster brain homogenate, subjected to serial rounds of PMCA, and subjected to western blotting.

FIG. 6 illustrates western blot assays of the samples. The numbers at the top of the gels indicate the number of days after inoculation. The numbers to the left of the gels indicate the number of PMCA rounds. The numbers at the bottom of the gels indicate the percentage of PrP^(Sc)-positive animals after four rounds of PMCA.

FIG. 7 illustrates plots of concentration versus the time period after inoculation for the various tissue and fluid samples. Endogenous replication of PrP^(Sc) reached high levels in spleens at early stages after infection (FIG. 7, plot A), which correlated with their presence in white blood cells (FIG. 7, plot C). Interestingly, PrP^(Sc) quantity decreased in spleens in the middle of the incubation periods, precisely prior to the time in which PrP^(Sc) began to appear in the brain (FIG. 7, plot B). The levels of PrP^(Sc) increased again in spleens close to the symptomatic phase, to reach a quantity similar to that found in the early pre-symptomatic stage of the disease (FIG. 7, plot A). The levels of PrP^(Sc) in brains increased in an exponential way with time, starting around 50 days post-inoculation (FIG. 7, plot B). PrP^(Sc) was not detectable in brains before this time, except for a few days after inoculation, which most likely represents the influx of PrP^(Sc) present in the inoculum across the blood brain barrier. The quantities of PrP^(Sc) estimated in brains two to nine days after inoculation reached around 2-4 fg/g of brain (FIG. 7, plot A). This quantity is probably not enough to trigger prion replication and is likely eliminated by the normal clearance mechanisms. The later re-appearance of PrP^(Sc) in the brains likely means a more constant influx of prions produced by peripheral replication and transport through the peripheral nerves. The biphasic behavior of PrP^(Sc) in spleens is similar to that expected in the blood buffy coat fraction, which mostly contains white cells. However, the quantities of PrP^(Sc) in buffy coat are three orders of magnitude lower than those measured in spleens (FIG. 7, plot C). In plasma, PrP^(Sc) was only detectable at or close to the symptomatic phase of the disease (FIG. 7, plot D), and the quantities are around 10 times lower than in the buffy coat fraction.

These findings indicate that the presence of PrP^(Sc) in blood may have two different sources: peripheral replication in the spleen at early stages and brain leakage at late stages. Prions in blood at the pre-symptomatic phase are restricted to the white cells, which likely were coming from cells previously resident in the spleen. At the symptomatic phase, cerebral prions are likely leaking to the blood and circulate in a cell-free manner in plasma and possibly produce a second wave of spleen infection.

A comparison of the estimated quantities of PrP^(Sc) in the organs and fluids tested at the symptomatic phase reveals that the quantity in the brain is 106, 108, and 109 times higher than in spleen, buffy coat, and plasma, respectively, in this particular model (Table 2). However, at half of the incubation period (50 days post inoculation) the quantity of prions in the brain is only around 2 fg/g, which represents only 3- and 2000-times higher than spleen and buffy coat (Table 2).

TABLE 2 Estimated PrP^(Sc) Concentrations (g/g tissue or g/mL of fluid) in Different Tissues and Biological Fluids at Distinct Time Periods After Inoculation Late Pre- Mid Pre- Early Pre- Symptomatic Symptomatic Symptomatic Symptomatic Source (110 dpi) (80 dpi) (51 dpi) (21 dpi) Brain  2.3 × 10⁻⁵ ± 6.8 × 5.1 × 10⁻¹¹ ± 4.8 × 2.2 × 10⁻¹⁵ ± 2.0 × Not detectable 10⁻⁶ 10⁻¹¹ 10⁻¹⁵ Spleen 2.0 × 10⁻¹¹ ± 1.1 × 5.2 × 10⁻¹³ ± 6.8 × 1.6 × 10⁻¹⁶ ± 1.2 × 8.0 × 10⁻¹² ± 7.1 × 10⁻¹¹ 10⁻¹³ 10⁻¹⁶ 10⁻¹² Buffy Coat 1.1 × 10⁻¹³ ± 0.9 × Not detectable 1.0 × 10⁻¹⁸ ± 1.0 × 1.9 × 10⁻¹⁸ ± 1.2 × 10⁻¹³ 10⁻¹⁸ 10⁻¹⁸ Plasma 5.2 × 10⁻¹⁵ ± 3.1 × Not detectable Not detectable Not detectable 10⁻¹⁵ Urine 2.0 × 10⁻¹⁶ ± 1.7 × Not done Not done Not done 10⁻¹⁶

To the extent that the term “includes” or “including” is used in the specification or the claims, it is intended to be inclusive in a manner similar to the term “comprising” as that term is interpreted when employed as a transitional word in a claim. Furthermore, to the extent that the term “or” is employed (e.g., A or B), it is intended to mean “A or B or both.” When the applicants intend to indicate “only A or B but not both” then the term “only A or B but not both” will be employed. Thus, use of the term “or” herein is the inclusive, and not the exclusive use. See, Bryan A. Garner, A Dictionary of Modern Legal Usage 624 (2d. Ed. 1995). Also, to the extent that the terms “in” or “into” are used in the specification or the claims, it is intended to additionally mean “on” or “onto.”

While the present application has been illustrated by the description of particular embodiments, and while the embodiments have been described in considerable detail, it is not an intention to restrict or in any way limit the scope of the appended claims to such detail. With the benefit of the present application, additional advantages and modifications will readily appear to those skilled in the art. Therefore, the application, in its broader aspects, is not limited to the specific details and illustrative examples shown and described. Accordingly, departures may be made from such details without departing from the spirit or scope of the general inventive concept. 

1. A method for preparing a calibration curve useful for estimating the concentration of PrP^(Sc) in a sample: preparing a plurality of stock solutions, each having a known concentration of PrP^(Sc); separately mixing each of the stock solutions with a first PrP^(C) source to form separate stock reaction mixes; performing a plurality of protein misfolding cyclic amplification cycles on the separate stock reaction mixes, each cycle comprising: incubating the stock reaction mix; and disrupting the stock reaction mix; subjecting the separate amplified stock reaction mixes to an assay after each cycle, until a PrP^(Sc) signal is detected; and comparing the concentration of each stock solution with the number of cycles required to detect the PrP^(Sc) signal.
 2. The method of claim 1, further comprising plotting the comparison in the form of a standard calibration curve.
 3. The method of claim 1, wherein the first PrP^(C) source is a normal tissue homogenate.
 4. The method of claim 1, wherein the assay is a western blot assay.
 5. A method for estimating the concentration of prion in a sample, the method comprising: mixing the sample with a non-pathogenic protein to form a reaction mix; performing a plurality of protein misfolding cyclic amplification cycles on the reaction mix, each cycle comprising: incubating the reaction mix; and disrupting the reaction mix; subjecting the amplified reaction mix to an assay after each cycle, until a prion signal is detected; and comparing the number of cycles required to detect the prion signal to a predetermined calibration curve.
 6. The method of claim 5, wherein the prion is PrP^(Sc).
 7. The method of claim 5, wherein the non-pathogenic protein is PrP^(C).
 8. The method of claim 5, wherein the disrupting comprises subjecting the reaction mix to sonication.
 9. The method of claim 5, wherein the assay is a western blot assay.
 10. The method of claim 5, further comprising: removing a portion of the reaction mix; contacting the portion with additional non-pathogenic protein to form a second reaction mix; performing a plurality of protein misfolding cyclic amplification cycles on the second reaction mix, each cycle comprising: incubating the second reaction mix; and disrupting the second reaction mix; subjecting the disrupted second reaction mix to an assay after each cycle, until the prion signal is detected; and comparing the number of cycles required to detect the prion signal to the predetermined calibration curve.
 11. The method of claim 5, wherein the sample is selected from: spleen, brain, blood, or urine.
 12. A kit for use in the method of claim 5, which comprises a known amount of non-pathogenic conformer and a calibration curve.
 13. A kit for detecting and quantifying prion in a sample, the kit comprising: a non-pathogenic protein; a sonicator; and a calibration curve.
 14. The kit of claim 13, wherein the prion is PrP^(Sc).
 15. The kit of claim 13, wherein the non-pathogenic protein is PrP^(C).
 16. The kit of claim 13, wherein the sample is selected from: brain, spleen, blood, or urine. 